Genomic alterations in plasma DNA from patients with metastasized prostate cancer receiving abiraterone or enzalutamide

Abstract

In patients with metastatic castrate-resistant prostate cancer (mCRPC), circulating tumor DNA (ctDNA) analysis offers novel opportunities for the development of non-invasive biomarkers informative of treatment response with novel agents targeting the androgen-receptor (AR) pathway, such as abiraterone or enzalutamide. However, the relationship between ctDNA abundance, detectable somatic genomic alterations and clinical progression of mCRPC remains unexplored. Our study aimed to investigate changes in plasma DNA during disease progression and their associations with clinical variables in mCRPC patients. We analyzed ctDNA in two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We conducted whole-genome sequencing (plasma-Seq) for genome-wide profiling of somatic copy number alterations and targeted sequencing of 31 prostate cancer-associated genes. The combination of plasma-Seq with targeted AR analyses identified prostate cancer-related genomic alterations in 16 of 25 (64%) treatment courses in the first cohort, in which we demonstrated that AR amplification does not always correlate with poor abiraterone and enzalutamide therapy outcome. As we observed a wide variability of ctDNA levels, we evaluated ctDNA levels and their association with clinical parameters and included the second, larger cohort for these analyses. Employing altogether 428 longitudinal plasma samples from 148 patients, we identified the presence of bone metastases, increased lactate dehydrogenase and prostate-specific antigen (PSA) as having the strongest association with high ctDNA levels. In summary, ctDNA alterations are observable in the majority of patients with mCRPC and may eventually be useful to guide clinical decision-making in this setting.

Keywords: androgen receptor; circulating tumor DNA; liquid biopsies; prostate cancer; targeted therapies.

Figure 1

Initial evaluation of somatic copy number alterations. (a) Workflow illustrating the processing of cohort 1 (USC). (b) Heat map of analyzed plasma samples after mFAST‐SeqS. Red indicates chromosome arms with increased z‐scores (>5) and blue chromosome arms with decreased z‐scores (≤5). Each row represents a chromosome arm (the short arms of the acrocentric chromosomes 13, 14, 15, 21 and 22 were omitted as they consist of repetitive DNA only), each column represents one plasma sample; the respective case identifiers are indicated below each column. (c) Circos plot illustrating the relative frequency of SCNAs (gains in red and losses in blue) of plasma samples with increased ctDNA levels (inner circle). The outer circle shows ideograms of the respective chromosomes. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Panel sequencing and pathway analyses. (a) Overview of somatic mutations (the pathogenic germline BRCA2 mutations were included, as they likely have a significant impact on the prostate cancer genome) as well as some SCNAs, i.e. deletions of the PTEN [10q23.31] and RB1 [13q14.2] loci, AR amplification and the TMPRSS2‐ERG rearrangement and pathways affected by these alterations. The oncoprint summarizes the 15 patients for which significant SCNAs and/or coding variants were detected. The eight columns (with the black symbols) to the right depict affected pathways (black and red numbers: patients treated with abiraterone and enzalutamide, respectively; blue number: patient treated sequentially with abiraterone followed by enzalutamide). (b) Plasma‐Seq profiles derived from patient #33572, whose tumor genome harbored two somatic mutations, i.e. L702H and T878A, in the AR gene (the x‐axis shows the chromosomes, the y‐axis indicates log2 copy number ratios; green: balanced regions; red: gained regions; blue: lost regions). While the baseline profile (33572‐42) was balanced, after 2 months with abiraterone treatment the progression profile (33572‐44) showed multiple SCNAs indicating increasing ctDNA, which was accompanied by raising ALP and PSA levels. The line graph displays PSA (brown) and ALP (blue) values obtained at the same time as the plasma sample (x‐axis: time points). (These color schemes for copy number profiles and ALP/PSA displays apply to all subsequent figures.) (c) Patient #28413 had a high‐amplitude AR amplification already at baseline. An increase in ctDNA was noted in the second plasma sample (28413‐63) with multiple SCNAs including the AR amplicon and PTEN loss at 10q23.31 (Log2‐ratio −0.98). [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Emergence of AR amplification during treatment with abiraterone or enzalutamide. (a) Patient #36976's baseline profile (36976‐37); at progression under therapy with abiraterone acetate plus prednisone and AR amplicon emergence 5 months later (36976‐25) and after therapy switch to enzalutamide another 5 months later (36976‐28). His maximal PSA decline was −38.1%. (b) Baseline and progression plasma‐Seq profiles of patient #37069 with emergence of AR amplification after 8 months and several SCNAs under treatment with enzalutamide (maximal PSA decline: −80.4%). [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Clinical significance of AR amplifications and of longitudinal tumor genome monitoring. (a) Plasma sample 34254‐53 from patient #34254, a BRCA2 germline mutation carrier, had several focal amplifications on the X chromosome, including one with the AR gene (the focal amplifications on the X chromosome were ChrX:8,051,929‐8,618,130; genes: MIR651, VCX2, VCX3B; ChrX:8,843,917‐9,070,658; gene: FAM9B; ChrX:36,926,063‐37,097,867; genes: FAM47C, FTH1P18; ChrX:46,087,026–47,558,082; gene: ARAF; ChrX:63,081,013–67,409,831; gene: AR; ChrX:82,358,796‐83,326,258; genes: POU3F4, CYLC1; ChrX:86,902,442‐90,681,961; genes: CPXCR1, TGIF2LX; ChrX:154,058,435‐154,346,865; genes: F8, FUNDC2, CMC4, MTCP1). Despite the AR amplicon, the patient responded for 20 months followed by only a slight PSA increase between 20 and 27 months and the ctDNA remained low (34254‐57). (b) Genome‐wide log2‐ratio plots of plasma samples from patient #35153. Between baseline (35153‐89) and progression (35153‐92) samples multiple novel relative copy‐number losses and gains occurred. Importantly, the AR amplification present in the first analyses (35153‐89 to 35153‐91) is lost in the last, i.e. 35153‐92, sample, as shown in the panels to the right. (c) The time interval between baseline (33763‐9) and progression (33763‐12) was 3–4 months and loss of the distal short arm of chromosome 1 and loss of the long arm of chromosome 16 were noted as novel changes. Progression of disease was also indicated by rising ALP and PSA values. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

Therapy response and PFS in relation to the z‐scores. (a) Waterfall plots indicating the maximal PSA response at weeks 9–14 for cases treated with abiraterone acetate plus prednisone (left) and enzalutamide (right). (b) Kaplan‐Meier plots of progression‐free survival for patients treated with abiraterone acetate plus prednisone and i‐score >5 and z‐score ≤5. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Predictors of ctDNA detection in metastatic CRPC. Violin plots of USC (a) and MUG (b) cohorts displaying the data densities of PSA, LDH, ALP and bone metastases values at each visit (the visit number color gradient is shown at the bottom). Table (c) displays the results of the univariate and multivariate analyses of ctDNA scores in relation to each of clinical covariates (bone metastases, LDH and PSA). [Color figure can be viewed at http://wileyonlinelibrary.com]